The aim of the present research proposal is to better understand the intermolecular contact regions of deoxyhemoglobin S (HbS) involved in the in vitro polymerization and to identify the functional groups in these regions amenable for chemical manipulation. Our studies to date have demonstrated the high selectivity of carboxyl groups of Glu-43(Beta) of HbS for derivatization during the carbodiimide activated coupling with glycine ethyl ester. We propose to continue these studies to target the modification to carboxylates at or near the intermolecular contact regions by manipulating the amine and/or carbodiimide components, and determine the influence of these derivatizations on the solubility, delay time, and other functional properties of HbS. N-(Beta-aminoethyl) 4-azido-2 nitroaniline, a photoactivable cross-linking reagent, will be introduced on specific carboxyl groups to study the topological changes around the intermolecular contact regions as the HbS molecule goes through the nucleation and the growth phases to form the gel. The reactivity of the guanidino groups of HbS toward phenylglyoxal will be studied, with the objective of introducing azidophenylglyoxal on specific arginine residue for chemical cross-linking studies. The potential usefulness of the latent cross-linking nature of glycolaldehyde and Alpha-hydroxy acetone in identifying the intermolecular contact regions of deoxy HbS involved in the nucleation and growth phases will be investigated. With the ultimate objective of understanding the interaction linkage of intermolecular contact regions of deoxy HbS during gelation, the fragment complementation studies that we have initiated will be continued. Procedures for the preparation of covalent analogs of Alpha- and Betas chains will be developed. New hybrid HbS with chemical mutations at two or more desired sites will be prepared to study the cooperativity of various intermolecular contact regions during polymerization. The results of these studies will lead to a better understanding of a) chemical reactivity of carboxyl and guanidino groups at the intermolecular contact regions and b) relative contribution of various intermolecular contact regions in the nucleation and growth phases of in vitro polymerization. The detailed knowledge of the structural aspects of polymerization of deoxy HbS is expected to lead to the design of new, specific reagents targeted to specific intermolecular contact regions. Such reagents that would neutralize the polymerizing influence of the mutation at Beta-6 position could pave the way for designing new antisickling agents.